ARTICLE pubs.acs.org/JAFC
Determination of 67Zn Distribution in Navy Bean (Phaseolus vulgaris L.) after Foliar Application of 67ZnLignosulfonates Using Isotope Pattern Deconvolution Ana Benedicto,†,§ Lourdes Hernandez-Apaolaza,† Inmaculada Rivas,‡ and Juan J. Lucena*,† †
Departamento de Química Agrícola and ‡Servicio Interdepartamental de Investigacion, Universidad Autonoma de Madrid, C/Francisco Tomas y Valiente 7, 28049 Madrid, Spain ABSTRACT: The improvement of Zn fertilizers requires new techniques to evaluate their efficacy. In this paper, the 67Zn stable isotope was used as tracer of several Znlignosulfonate complexes to study the foliar-applied Zn uptake and distribution behavior in the plant, compared with ZnEDTA. Navy bean plants (Phaseolus vulgaris L.) were grown hydroponically in a Zn-free nutrient solution, and six modified lignosulfonates and EDTA complexed with 67Zn were used in foliar application in the young leaves as Zn sources. Zinc isotopes in roots, stems, and sprayed and unsprayed leaves were determined by ICP-MS, and signal interferences caused by the compounds of the digested vegetal samples were corrected. The mathematical procedure of isotope pattern deconvolution allowed the minimization of the uncertainty in the measured molar fractions of Zn from fertilizer or from natural sources. Significant differences in Zn use and distribution were observed among the fertilizers when the calculated concentrations of Zn from the fertilizer were compared, whereas they were unnoticeable attending to the total Zn in plant tissues, usually determined at the conventional studies. By foliar spray, higher Zn uptake and mobilization to leaves and stems were achieved with 67ZnEDTA than with 67ZnLS complexes. The ultrafiltered LS and phenolated LS showed slightly better ability to provide Zn to the bean plants than the other LS. The foliar-applied Zn use and distribution in the plant were related with the stability of the Znlignosulfonates complexes. Those presenting the lower stability versus pH, but the highest complexing capacity, were slightly more suitable to supply foliar-applied Zn to navy beans. KEYWORDS: 67Zn, lignosulfonates, fertilizers, foliar application, isotope pattern deconvolution
’ INTRODUCTION Zinc deficiency is a widespread micronutrient disorder among different crops1,2 affecting either the quantity or the nutritional quality of the harvest.3 This fact becomes especially important because inadequate dietary Zn in a population has a high prevalence in the world (estimated at 31% in global population). Moreover, the risks of morbidity and mortality associated with Zn deficiency are relatively high.4 Consequently, special efforts have been made in the study of Zn fertilization of crops. Traditional fertilizer experiments are not able to distinguish between the Zn applied with the fertilizer and the background Zn level in plant tissues. However, the incorporation of an isotopic tracer allows a detailed knowledge of the uptake, transport, and accumulation of this element in plants. High precision and low detection thresholds reached with isotopic tracer techniques could allow finding differences normally not detected with conventional techniques. This fact is highly relevant in the comparison of new fertilizers, for which differences may not be assessed by using the traditional techniques, as the determination of total Zn in plant tissues. In the literature, there are several studies that use the radioactive isotope 65Zn to evaluate the efficacy of Zn fertilizers512 and the Zn availability in soils.13 However, the development of sensitive analytical techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), allows also the use of stable isotopes for this application. The use of stable instead of radioactive isotopes gives a high flexibility in the experimental designs used and can include field studies, because special safety measurements and trained staff are not required. r 2011 American Chemical Society
Moreover, long-term assays can be carried out with no radioactivity decay over time. In addition, the generation of radioactive wastes is avoided. For this purpose, the stable isotope 68Zn has been used in several studies to evaluate absorption and translocation of Zn in plants.1417 Also, 67Zn, another stable isotope, should be considered. 67Zn natural abundance (4.1%) is around 4 times lower than that of 68Zn (18.75%), so a greater efficacy of the former as tracer could be expected. The stable isotope 67Zn was previously used to track the amount of Zn provided by rainfall or acid fog in a forest;18 however, as far as we know, this isotope has not been used as a tool to study the Zn nutrition in plants. Accuracy and precision of stable isotope ratio measurements by ICP-MS in plant tissues require the correction of possible interferences and of the instrumental mass bias. For example, to determine 57Fe in a plant matrix by ICP-MS, RodríguezCastrillon et al.19 carried out the internal mass bias correction by using an isotope pattern deconvolution mathematical procedure as a technique for isolating distinct isotope signatures from mixtures of natural abundance and enriched tracers. It was observed that, for plants with low 57Fe enrichment, isotope pattern deconvolution achieved lower tracer/tracee ratio uncertainties than the traditional methods, which permitted a more accurate assessment of the Fe from different sources. Received: January 20, 2011 Revised: June 28, 2011 Accepted: July 7, 2011 Published: July 07, 2011 8829
dx.doi.org/10.1021/jf2002574 | J. Agric. Food Chem. 2011, 59, 8829–8838
Journal of Agricultural and Food Chemistry Table 1. Instrument Conditions ICP-MS Elan 6000 ICP RF power
1200 W
nebulizer gas flow
0.78 L min1
sampler/skimmer cone
Ni
lens voltage
9.50 V
analogue stage voltage pulse stage voltage
2420 V 1550 V
sweeps/reading
20
readings/replicate
1
replicates
3
scanning mode
peak hopping
dwell time
50 ms per point
rinse time
45 s 48 min1
sample uptake time read delay
30 s 48 min1 30 s 20 min1
Several Znlignosulfonates (ZnLS) with different physicalchemical characteristics are the fertilizers tested in this study, compared with the synthetic chelate ZnEDTA. Lignosulfonates are byproducts obtained from the pulp and paper industry used as complexing agents in >50% of the commercial Zn formulations available in the Spanish market in 2009.20 Application of LS in the formulation of Zn fertilizers implies the reuse of the black liquor obtained in these industries, which is potentially a pollutant material. These Zn natural organic complexes are a more ecocompatible and less expensive alternative to the synthetic chelates. However, they are in many cases less effective than chelates because of the weak bonds between the metal and the complexing agent, which determines the low stability of these products.21 The understanding of the relationship between their physicalchemical characteristics and their efficacy in plants will be decisive in choosing among LS from different types of wood available in the market and the most suitable modification processes required to improve their efficacy as fertilizers. The aim of this study is to test the suitability of 67Zn as a tool to compare the Zn uptake and distribution behavior in navy beans after the foliar application of six ZnLS. The isotope was quantified by ICP-MS, the data were processed using a novel isotope pattern deconvolution mathematical procedure, and mass bias correction was performed internally. Interference studies and Zn isotope ratio measurements were also carried out in plant tissue digested samples.
’ MATERIALS AND METHODS Zinc Determinations in Plant Tissues by ICP-MS and Isotope Pattern Deconvolution. Instrumentation. The instrument used was an ELAN 6000 quadrupole ICP-MS Perkin-Elmer Sciex. The sample introduction system consisted of a Ryton cross-flow nebulizer with a Ryton double-pass Scott spray chamber. Sample transport from the autosampler to the nebulizer was established by a peristaltic pump. Instrumental settings used for the analysis are summarized in Table 1. All standard and sample solutions were prepared by using water according to EN ISO 3696,22 grade I, free of organic contaminants and HNO3, Suprapur (Merck). Daily verification of the instrument was performed by using a 10 μg Li, Mg, Rh, Ba, Ce, and Pb L1 mixture in 1% (w/w) HNO3 solution to check the correct instrument response; the oxides (CeO) and double-charge ions (Ba2+) formation levels were
